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Title Nicotine does not reduce Nosema ceranae infection in honey bees

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Journal INSECTES SOCIAUX, 67(2)

ISSN 0020-1812

Authors Hendriksma, HP Bain, JA Nguyen, N et al.

Publication Date 2020-05-01

DOI 10.1007/s00040-020-00758-5

Peer reviewed

eScholarship.org Powered by the California Digital Library University of California Nicotine does not reduce Nosema ceranae infection in honey bees

H. P. Hendriksma, J. A. Bain, N. Nguyen & J. C. Nieh

Insectes Sociaux International Journal for the Study of Social Arthropods

ISSN 0020-1812

Insect. Soc. DOI 10.1007/s00040-020-00758-5

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Insectes Sociaux https://doi.org/10.1007/s00040-020-00758-5 Insectes Sociaux

RESEARCH ARTICLE

Nicotine does not reduce Nosema ceranae infection in honey bees

H. P. Hendriksma1,2 · J. A. Bain1 · N. Nguyen1 · J. C. Nieh1

Received: 26 January 2020 / Revised: 26 January 2020 / Accepted: 18 February 2020 © International Union for the Study of Social Insects (IUSSI) 2020

Abstract Bee-collected pollen and nectar contain multiple phytochemicals that can have anti-pathogenic efects when ingested. For example, the plant alkaloid, nicotine, can reduce infections by the trypanosome gut parasite (Crithidia bombi) in bumblebees. Parasitized bumblebees may be drawn to nicotine and thereby self-medicate their infection. We tested the hypothesis that nicotine can reduce infections of a common microsporidian pathogen, Nosema ceranae, in the honey bee gut. We found, however, that that a feld realistic exposure dose of 1 ppm nicotine was not preferentially consumed by Apis mellifera foragers fed live Nosema spores (5 × 104 spores per bee; N = 160). One-day-old bees infected with Nosema (1 × 104 spores per bee; N = 160) showed no repression of nosemosis over a chronically applied exposure gradient of 0, ­10−2, ­10−1, ­100, ­101, ­102, ­103 or ­104 ppm nicotine. Since imbibed nicotine may not efectively reach the spores in the bee gut, we conducted an in vitro experiment, in which Nosema spores were exposed up to 10­ 4 ppm nicotine in vials, rinsed of nicotine, and then fed to 1 day old bees (2 × 104 spores per bee; N = 216). However, the in vitro nicotine-treated spores remained infectious. Nicotine did impair bee mortality at high concentrations. Dietary nicotine is evidently not a treatment for nosemosis, but future studies should continue to examine the role of phytochemicals and bee health.

Keywords Apis mellifera · Host–parasite interaction · Intracellular parasite · Nectar · Preference · Self-medication

Introduction of approximately 500,000 spores per infected bee (Rennich et al. 2012). Infection with Nosema ceranae is a common The Western honey bee, Apis mellifera, is one of the most factor that likely contributes to observed colony losses in the abundant pollinator species in both natural and agricul- USA (Cox-Foster et al. 2007). tural habitats, but, in many regions, honey bees have poor There are two common microsporidian Nosema species health (Garibaldi et al. 2013; Hung et al. 2018). In regions that infect honey bees: Nosema apis (Zander 1909) and of Europe and the USA, honey bee colonies have shown Nosema ceranae (Fries et al. 1996). Nosema infects the declines due to a variety of stressors, including the spread epithelial cells of the midgut of honey bee adults (Webster of diseases (Goulson et al. 2015; Moritz and Erler 2016). 1993) and larvae (Eiri et al. 2015). Nosema ceranae is native A honey bee disease survey found that approximately 70% to Eastern honey bee colonies (Apis cerana) but has rap- of managed colonies in the USA are annually infected idly spread since the 1990s to A. mellifera colonies around with the microsporidian pathogen Nosema, at an average the world and has evidently largely displaced N. apis (Klee et al. 2007; Papini et al. 2017; Sinpoo et al. 2018). Bees can be treated with the antibiotic fumagillin, which inhibits the Electronic supplementary material The online version of this enzyme methionine aminopeptidase-2 and thereby disrupts article (https​://doi.org/10.1007/s0004​0-020-00758​-5) contains supplementary material, which is available to authorized users. spore protein maturation (Sin et al. 1997; Huang et al. 2013). However, multiple countries prohibit fumagillin use because * H. P. Hendriksma of the risk of residues in honey. In addition, the repeated use [email protected] of this antibiotic may have contributed to parasite resistance, Nosema 1 Division of Biological Sciences, Section of Ecology, thus reducing options for treating infections (Tian Behavior, and Evolution, University of California San Diego, et al. 2012; Huang et al. 2013). Identifying novel and efec- 9500 Gilman Drive, MC0116, La Jolla, CA 92093, USA tive treatments for Nosemosis is therefore critical. 2 Present Address: Institute for Bee Protection, Julius Kühn-Institut, Messeweg 11/12, 38104 Brunswick, Germany

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H. P. Hendriksma et al.

Although N. ceranae infection does not typically lead to 2015; Thorburn et al. 2015). Bernklau et al. (2019) recently colony death, it can weaken colonies and has synergistic demonstrated that cafeine, kaempferol, and p-coumaric efects with pesticide exposure and other causes of poor acid (25 ppm) could reduce N. ceranae spore loads in honey colony health (Pettis et al. 2012). Nosema can reduce vitel- bees, increasing the longevity of infected bees. Cafeine and logenin (Vg) and increase Juvenile Hormone (JH) titers in nicotine are chemically similar alkaloids with relatively low bees—the reverse of what is normal for healthy individuals molecular masses, having a carbon ring and one or more of nursing age (Goblirsch et al. 2013). Because the transi- nitrogen atoms. Like cafeine, nicotine may be a suitable tion from nursing to foraging is regulated by the interaction plant compound to use against N. ceranae infection in honey of Vg and JH, young Nosema-infected bees begin to forage bees (Köhler et al. 2012; Baracchi et al. 2017). precociously, an atypical behavior (Paxton 2010). Nosema Nicotine is commonly found in pollen and nectar of Sola- can thus contribute to the decline of brood care by nurses, naceae, a family of fowering plants with a worldwide dis- the premature death of foragers, and poorer overall colony tribution, which includes food and medicinal plants. Some condition and health (Goblirsch et al. 2013). members of this family are very attractive to honey bees Multiple plants produce secondary compounds that can because they provide easily accessible inforescences and be benefcial to animals. These secondary compounds in copious pollen and nectar: morning glory, jimsonweed, and nectar are generally viewed as a byproduct of plant defensive sweet potato (Crane et al. 1984). Other members of this fam- strategies against herbivores (Stevenson et al. 2017), yet may ily depend upon buzz-pollination by bees: tomatoes, egg- also infuence pollinators (Wright et al. 2013). Preferential plant, bell and chili peppers (Buchmann 1983). Tobacco collection of benefcial phytochemicals in pollen and nec- plants (Nicotinia tabacum) can be a source of high honey tar by pollinators could even be a self-medication behavior production for honey bee colonies, with 40 kg/colony/season (Erler and Moritz 2016). For example, some insects can reported in the USA (Espina Perez and Ordetx Ros 1983). reduce parasite infections by consuming secondary com- Bees can, therefore, be exposed to nicotine via pollen and pounds from plants. Woolly bear caterpillars infected with nectar. lethal endoparasitic larvae of tachinid fies ingested more We hypothesized that nectar with nicotine is parasiticidal parasiticidal alkaloids than unparasitized individuals (Singer to N. ceranae in A. mellifera, and that infected bees may et al. 2009). Fruit fies (Drosophila melanogaster) consumed self-medicate by preferring nicotine laced sucrose solution alcohol to reduce infections by endoparasitoid wasps (Milan to pure sucrose solution. We asked fve research questions et al. 2012). (Fig. 1). Q1: At what concentration does nicotine afect Honey bee colonies collect plant resins, creating propo- honey bee survival? Q2: Does Nosema-infection change lis (Burdock 1998; Bankova et al. 2019) that they use to sucrose consumption by honey bees? Q3: Do parasitized seal and strengthen the nest. Propolis also has antibacte- honey bees prefer food with nicotine more than food without rial, antifungal and antiviral activity (Freitas et al. 2019) nicotine? Q4: Can N. ceranae be suppressed within honey and is potentially produced in response to colony infec- bees via nicotine consumption? Q5: Is nicotine parasiticidal tions, to maintain colony health, or both (Simone-Finstrom to N. ceranae spores outside the host? and Spivak 2012; Gherman et al. 2014; Erler and Moritz 2016). Propolis may also help to suppress Nosema infec- tion (Gherman et al. 2012; Yemor 2016), and other natu- Materials and methods ral products may also help. Sunfower pollen collected by honey bee colonies reportedly reduced Nosema infections Honey bees (Giacomini et al. 2018). Nicotine and other allelochemicals can suppress proliferation of the trypanosome gut parasite, We used honey bee colonies (Apis mellifera ligustica) kept Crithidia bombi, in bumblebees (Manson et al. 2010; Biller at an apiary at the Biological Field Station at UC San Diego et al. 2015; Palmer-Young et al. 2017a; Richardson et al. in La Jolla, California (32° 53′ 13 N, 117° 13′ 49 W). The

Fig. 1 Schematic overview of research questions. Potential interactions between a parasite (Nosema), its host (a honey bee), and a dietary phytochemi- cal (nicotine) are listed in the Venn diagram with our fve research questions shown to the right

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Nicotine does not reduce Nosema ceranae infection in honey bees colonies were managed following normal beekeeping prac- (Gisder and Genersch 2013), and amplicon sequencing and tices. All colonies were queenright and healthy, as deter- confirmed via GenBank sequence comparison that the mined by standard inspection techniques. Diferent colonies spores were N. ceranae. were used for diferent experiments (see below). Bees were incubated (Nor-Lake Scientifc, model LRI- Q1: Nicotine and dimethoate toxicity to honey bees 201WWW/0) under dark conditions in cages in the lab (see below). Test Q1 was performed at 24.7 ± 1.4 °C (mean ± SD) We analyzed bee survival to calculate nicotine and with a Relative Humidity (RH) of 81.5 ± 7.7%, as constantly dimethoate ­LD50 values at 24 h and 48 h after feeding. monitored with data loggers (Onset HOBO, Wilmington, Dimethoate is an organophosphate insecticide and is a stand- NC). Tests Q2 trough Q5 were performed at 34.2 ± 0.2 °C ard reference toxin used to validate bioassays (Medrzycki with a of 65.1 ± 5.4%. et al. 2013). The ­LD50 is the amount of ingested substance that kill 50% of a test population within a specifed time Nosema spores period. This toxicity value can be based on acute (one-time) exposure or chronic (constant) exposure and is commonly Nosema ceranae spores came from a stock of infected expressed in µg/bee for honey bees, which is an alternative bees continuously renewed and maintained in bees kept to the expression in mg/kg body weight (Johnson 2015). Our in an incubator. Spores were collected by dissecting mid- toxicity tests followed the guidelines of the European and guts from ten infected bees, and homogenizing the guts in Mediterranean Plant Protection Organization (EPPO) and 100 µl deionized (DI) water in a 1.5 ml microcentrifuge tube the International Commission for Plant–Pollinator Relation- using a Kontes electric pestle. Homogenates were strained ships (ICPPR). Specifcally, we used the OECD guidelines with a Buchner funnel and two flter papers with 2.5 µm for the acute oral toxicity tests on honey bees (OECD 1998; pore size (Grade 5; Whatman) into a 100 ml Erlenmeyer Medrzycki et al. 2013). fask under vacuum. The fltrate was transferred into 1.5 ml We designed nicotine test concentrations based upon 4 microcentrifuge tubes and centrifuged at ­10 rpm (6702g) Detzel and Wink (1993) who report an oral 48 h LD­ 50 of for 6 min (Eppendorf 5415D Centrifuge). After discarding 0.2% (2 × 103 ppm nicotine), for honey bees chronically fed supernatants, the spore pellets were combined into one spore nicotine. Test solutions were prepared using DI water with concentrate with a volume of 0.5 ml. Spore concentrations 1.5 M analytical-grade sucrose (Fisher Scientifc, Fair Lawn, were measured with a Neubauer Improved Hemocytometer NJ). Liquid (−)-Nicotine was obtained from Sigma-Aldrich on a Zeiss Microscope under 400× magnifcation (Cantwell (N3875-5ML; Milwaukee, WI, USA). For dimethoate toxic- 1970). Dissection, extraction, and concentration of fresh ity testing we used a concentration range based upon an acute spores occurred on the same day as the choice trial (§2.4) oral 24 h ­LD50 for honey bees of 0.10–0.35 μg dimethoate and the in vitro trials (§2.6). However, for the in vivo trial (OECD 1998). Dimethoate was obtained from Fluka Chemie (§2.5) we used a stored spore stock that had been refriger- (Switzerland). All test levels were in a geometric concentra- ated for 3 weeks at 4 °C. To determine spore identity, we tion series with spacing factor of two (Table 1). used standard DNA extraction methods, PCR amplifcation Brood combs without worker bees were incubated for with the primer pair NoscRNAPol-F2 and NoscRNAPol-R2 20 h in a nuc box at 33 °C and 70% RH. The emerged bees

Table 1 LD50 experimental Formulation Concentration Dose/bee design 10 µl nicotine + 7990 µl sucrose soln. 0.25 µl/200 µl (1250 ppm) 12.6 µg 10 µl nicotine + 3990 µl sucrose soln. 0.50 µl/200 µl (2500 ppm) 25.3 µg 25 µl nicotine + 4975 µl sucrose soln. 1.00 µl/200 µl (5000 ppm) 50.5 µg 25 µl nicotine + 2475 µl sucrose soln. 2.00 µl/200 µl (10,000 ppm) 101.0 µg 50 µl nicotine + 2450 µl sucrose soln. 4.00 µl/200 µl (20,000 ppm) 202.0 µg 16.14 mg dimethoate + 10 ml sucrose soln.; 1:800 0.4035 µg/200 µl (2 ppm) 0.0202 µg 16.14 mg dimethoate + 10 ml sucrose soln.; 1:400 0.8070 µg/200 µl (4 ppm) 0.0404 µg 16.14 mg dimethoate + 10 ml sucrose soln.; 1:200 1.6140 µg/200 µl (8 ppm) 0.0807 µg 16.14 mg dimethoate + 10 ml sucrose soln.; 1:100 3.2280 µg/200 µl (16 ppm) 0.1614 µg 16.14 mg dimethoate + 10 ml sucrose soln.; 1:50 6.4560 µg/200 µl (32 ppm) 0.3228 µg 50 g sucrose + 50 g DI water 50% w/v sucrose 0 (control)

The acute oral toxicity of nicotine and dimethoate on honey bees was tested with the following doses. The density of 1.01 g/cm3 nicotine was used to convert 10 µl exposure volumes per bee to mg doses

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H. P. Hendriksma et al. were collected in four plastic cages (500 cm3) and ofered spores per cage; mean ­104 spores per bee), the consump- solutions of 50% w/v sucrose solution, ad libitum. All bees tion and mortality of the bees were recorded over 14 days. were 14 days old when they were tested, the average age at Bees were fed nicotine solutions chronically with a diferent which honey bees begin foraging (Schippers et al. 2006). concentration for every cage. A stock solution of ­104 ppm 2 h before the test, bees were transferred into 36 dispos- (1 mg/l) nicotine was made in a 50% sucrose solution (w/v) able 80 cm3 paper test cages, with a ventilated bottom and prepared with DI water. Subsequently we made 1:10 serial a clear plastic sheet at the front. Eleven treatment doses dilutions ­(104, ­103, ­102, ­101, ­100, ­10−1, ­10−2 ppm). After (Table 1) were replicated with four colonies and 20 bees per 14 days of chronic exposure, Nosema infection in bees was cage (11 × 4 × 20 = 880 bees). Each cage contained 200 µl assessed for N = 72 midgut samples, with spore counts per- of the solution in the caps of two 1.5 ml microfuge tubes, formed in duplicate and the average calculated per bee. thus providing 10 µl solution per bee, on average. The bees consumed all test solutions within 4 h. The emptied caps Q5: Nosema exposure to high nicotine concentrations, were then removed and two 5 ml syringes with 50% (w/v) in vitro pure sucrose solution were introduced. Each syringe had the tip cut of to create a 2 mm diameter opening for feeding. Freshly collected Nosema spores were exposed to nicotine Sucrose solution was provided ad libitum. Mortality was concentrations in vitro, then washed, and subsequently fed to recorded daily over 4 days. bees to determine their ability to infect bees. For example, a 2 × 104 ppm nicotine solution was made with DI water, then Q2 and Q3: Nosema infection, nicotine choice and sucrose mixed with an equal volume of fresh Nosema spore extract, consumption by bees thus creating a ­104 ppm nicotine/spore mix. The mixtures were incubated at room-temperature (21 °C) for 60 min at Forager bees were collected upon their return to their respec- 300 rpm (Eppendorf Thermomixer 5350). After incuba- tive hives for preferential nicotine consumption tests. We tion, spores were spun down into a pellet by centrifugation targeted foragers because they can choose what to bring back for 10 min at 12 × 103 rpm (16,128g, a higher speed than to the hive, unlike young hive-bees. The foragers were kept our initial purifcation method to ensure that we pelleted as in 16 paper cages (160 bees of four colonies; ten bees per many of the treated spores as possible). Supernatants were cage). All bees were experimentally infected with Nosema carefully removed and pellets were resuspended in 1 ml DI spores by feeding 5 × 105 spores per cage (5 × 104 per bee). water to rinse the spores. Spores were then re-pelleted in the All cages were given two feeding syringes with 50% (w/v) centrifuge following the prior steps to discard the superna- sucrose solution; one with nicotine at 1 ppm, and the other tant water. The rinsed spores were resuspended in 50% w/v without. Solution consumption was measured by weighing sucrose solution so that 7 µl of solution contained approxi- all syringes every second day. The positions of the control mately 2 × 104 spores (Table S1). syringe and nicotine treatment syringe were swapped after Test bees were collected within 24 h of emergence from weighing, to counter potential spatial bias efects within brood combs (N = 6 colonies) and incubated overnight at cages. Syringes and solutions were all replaced at day 7. 34 °C. Each bee was individually fed the pre-treated spores Evaporation under test conditions was measured with refrac- by inserting a pipette tip with 7 µl solution through the lid of tometry: the solutions were 43.6°Bx at day 0 (50% w/v), a 13 ml clear styrene snap cap vial (23 mm diameter × 44 mm yet 51.0°Bx at day 5, indicating a daily evaporation loss of high). The vials were placed on trays in a dark incubator at mean 1.8 ± 0.4% (N = 20 syringes). We did not apply a data 34 °C. Approximately 50% of bees had consumed their dose correction for evaporation since solutions with and without in 1 h, and 95% of bees had imbibed the dose within 4 h. We nicotine did not difer in evaporation loss (2-sided t test: discarded bees that had not fully consumed their full dose t = 0.53, p = 0.40). Bee survival was recorded daily, and dead (fuid still visible in the pipette tip). After exposure, ten bees bees were removed. Per cage (N = 16), one bee was randomly were grouped per treatment and colony in 500 cm3 plastic collected at day 14 to verify successful Nosema infection cages. Each cage, with ventilation and feeding holes, was (Cantwell 1970). equipped with two feeding syringes with 50% (w/v) sucrose solution. After 14 days, all bees were frozen at − 20 °C to Q4: Nosema virulence over a nicotine gradient fed to bees assess infection results via hemocytometer spore counting in vivo (Cantwell 1970). Each spore pre-treatment batch was replicated six times We tested if nicotine could reduce Nosema infection when (Table 2). Every spore feeding concentration was checked fed to bees (Nbees = 160, Npaper cages = 8, Ncolony = 1). In this (N = 18; Table S1). A fumagillin positive control treatment in vivo experiment, we fed all bees with spores. After at 25 ppm followed the recommended concentration for Nosema spores were fed to newly emerged bees (2 × 105 colony treatment (Huang et al. 2013; Palmer-Young et al.

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Nicotine does not reduce Nosema ceranae infection in honey bees

Table 2 Nosema spore pre-treatment with nicotine were made with Tukey’s Honestly Signifcant Diference Honey bee infection with Nosema (HSD) tests (Fig. 3). For the in vivo experiment, sucrose consumption and No Nosema spores (control 1) 48 h bee survival per cage (N = 8) were correlated with lin- Spores pre-exposed to Fumagillin (control 2) ear regression (Fig. 4a, b). Midgut spore counts between Spores pre-exposed to DI water (0 ppm) nicotine levels were compared with a non-parametric test Spores pre-exposed to 1 ppm nicotine (Kruskal–Wallis). We made pairwise comparisons with 2 Spores pre-exposed to 10­ ppm nicotine Mann–Whitney tests and Dunn’s Multiple Comparison 4 Spores pre-exposed to 10­ ppm nicotine tests. We performed a linear regression over the fve nico- To assess if nicotine is parasiticidal in vitro, Nosema virulence in tine concentration levels (0 ppm excluded) versus the midgut bees was tested after a spore pre-treatment with nicotine Nosema spore counts (Fig. 4c). The efect of pre-treating Nosema spores in vitro, before honey bee infection with nicotine, fumagilin or DI water, 2017b). In one of our six colonies, we found Nosema infec- was analyzed with Generalized Mixed Efects models. The tion in the negative control bees and therefor this colony was response variable, midgut spores 14 days post-infection, removed from the analysis (Table S1). were ­log10 transformed (count + 1) and analyzed with treat- ment as an explanatory variable (six levels), and colony (fve Statistical analyses levels) nested in replicate trials (two levels) as a random factor. Due to our rinsing protocol, bees were fed slightly Data analyses were performed with JMP Pro 13.1.0 soft- diferent amounts of spores in this experiment. We, there- ware. Probit analyses were used to calculate 24 h and 48 h fore, tested for an efect of the number of spores fed to bees LD­ 50 values. Measures of uncertainty were estimated with but removed this variable because it was not signifcant (see SE, and measure diferences were indicated with 95% con- Table S1). Pairwise post hoc comparisons were made with fdence intervals (Finney 1952). A Schneider-Orelli (1947) Tukey’s HSD tests (Fig. 5). adjustment for background mortality was applied. We report our results as mean ± standard error. Forager consumption and choice among feeding solu- Results tions, with or without 1 ppm nicotine, after experimental Nosema infection, were analyzed with Linear Mixed Efects Nicotine toxicity to honey bees (Q1) The acute oral 48 h Models. Daily solution consumption was analyzed as the LD­ 50 dose was 80.5 ± 22.7 µg nicotine per bee, and the response variable, with nicotine (two levels; 0 and 1 ppm) organophosphate insecticide dimethoate was 2000 times and colony background (four levels) as fxed factors, and more toxic at a dose of 0.041 ± 0.014 µg/bee (Fig. 2, time as a covariate (11 time points). Following standard Table 3). The background mortality was 0% at 24 h, 5% procedures, we frst ran models with nicotine × time and at 48 h, 8.8% at 96 h, for N = 80 unexposed bees. The con- nicotine × colony interactions but removed the interactions fdence intervals indicated that the 24-h and 48-h toxicity if they were not signifcant. Pairwise post hoc comparisons values did not signifcantly difer (Table 3).

Fig. 2 Survival of bees after exposure to diferent concentrations of nicotine (a), and diferent concentrations of dimethoate as a positive toxicity control (b). These survival data were used to calculate the fnal 24 h and 48 h ­LD50 values (Table 3)

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Table 3 Acute oral toxicity LD Nicotine (Fig. 2a; N = 480 bees) Dimethoate (Fig. 2b; N = 480 bees) of nicotine and dimethoate to 50 honey bees 24 h 65.2 µg, 95% CI [46.0–92.3], N = 24 cages 0.053 µg, 95% CI [0.037–0.077], N = 24 cages Mean 78.7 µg ± 23.6 SE, N = 4 colonies Mean 0.053 µg ± 0.019 SE, N = 4 colonies 48 h 78.6 µg, 95% CI [53.5–115.4], N = 24 cages 0.098 µg, 95% CI [0.072–0.135], N = 24 cages Mean 80.5 µg ± 22.7 SE, N = 4 colonies Mean 0.041 µg ± 0.014 SE, N = 4 colonies

Table 4 Overview of honey bee experiments with experimental Nosema infection Nosema trials Nicotine exposure (ppm) Bee age Spores fed Mean spores (million) Amplifcation

Choice, §2.4 0 and 1 Forager 50 × 103/bee 7.15 ± 2.47 SE, N = 16 ×142 In vivo, §2.5 0, ­10−2,10−1,100,101,102,103,104 0–14 days 10 × 103/bee 1.93 ± 0.21 SE, N = 72 ×204 In vitro R1, §2.6 0, 1, ­102, ­104 0–14 days 20 × 103/bee 6.28 ± 0.97 SE, N = 36 ×315 In vitro R2, §2.6 0, 1, ­102, ­104 0–14 days 14 × 103/bee 1.52 ± 0.20 SE, N = 36 ×107

Fig. 3 Consumption and choice of infected honey bee foragers feeding on sucrose with and without nicotine. Shown are (a) the mean sucrose solution consumption in mg per bee per day with SE error bars for N = 4 colonies, and (b) means with SE error bars over N = 11 time- points. Diferent letters indicate signifcant diferences (Tukey’s HSD Test, p < 0.05)

Fig. 4 Honey bee infection with Nosema, with in vivo exposure to Median and quartile spore counts are shown by boxplots, with error nicotine. Nosema parasitized bees, in eight cages, were chronically whiskers indicating minimum and maximum values for N = 72 mid- exposed to nicotine over a ­107 fold increasing concentration range gut spore counts. Diferent letters indicate signifcant diferences for 14 days. a Bees exposed to nicotine doses higher than 10 ppm between treatments (Dunn’s Multiple Comparison Test, p < 0.05). No reduced their consumption (grey dashed line). b Survival decreased spore counts for nicotine doses 1000 and 10,000 ppm due to bee mor- sharply for nicotine concentrations > 100 ppm (dashed black line). c tality

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Nicotine does not reduce Nosema ceranae infection in honey bees

p = 0.04), however, the Nosema spore count did not signif- cantly increase or decrease over the fve nicotine dose levels (regression; p > 0.05). Nicotine and Nosema in vitro (Q5) Honey bees can metabolize ingested nicotine (Du Rand et al. 2017a, b), thus nicotine may not reach the sites of Nosema infection. We, therefore, also tested the efect of pre-treating Nosema with nicotine in vitro (outside of the honey bee host), a method that also enabled us to test supra-lethal concentrations. Over 97% of bees fed the pre-treated Nosema spores were success- fully infected (2.94 million midgut spores on average), which was signifcantly diferent from the negative control group in which 0% of bees were infected (F5,80 = 87.3, p < 0.001; Tukey HSD test, p < 0.05). Interestingly, pre-treated spores that had been exposed to water (0 ppm nicotine), or fumagil- lin, were just as infective as spores that had been exposed to 1, ­102 or ­104 ppm nicotine (Fig. 5).

Discussion Fig. 5 Nosema spore pre-treatment, in vitro. The boxplots show medians and quartiles of Nosema spore counts, 14 days post infec- Multiple animals can use natural compounds such as alka- tion, of 108 honey bee midguts (Table S1). Error whiskers indicate loids to counter pathogen infections. We tested the hypoth- minimum and maximum values with outlier data points as dots. Diferent letters indicate a signifcant diference (Tukey HSD test, esis that the alkaloid, nicotine, can help reduce infections p < 0.05) of the common gut parasite N. ceranae in the honey bee, A. mellifera. However, Nosema infection levels were not afected when we fed nicotine to bees directly, or frst pre- Nosema and nicotine consumption by honey bees (Q3) treated spores with nicotine and then fed these treated spores Our Nosema spores did infect bees. Midgut spore counts to bees—despite testing a wide range of nicotine concen- showed that bees fed spores had 107- to 315-fold more spores trations that ranged from feld realistic (1 ppm) to fatal by the end of the experiment than they were fed (Table 4). ­(104 ppm; 100% bee mortality). Bees infected with Nosema did not signifcantly consume With an acute oral 48 h-LD50 of 53.5–115.4 µg (95% more 1 ppm nicotine sucrose solution than the 0 ppm nico- CI) per honey bee, nicotine is classifed as essentially non- tine sucrose solution (Fig. 3; F1,328 = 0.05, p = 0.82). Con- toxic to bees (Environmental Protection Agency guide- sumption did vary based on colony background: colony 2 lines, LD­ 50 > 11 µg/bee). Following 48 h of chronic expo- consumed signifcantly more than other colonies (Fig. 3a; sure (Fig. 4b), our median bee survival at ­103 and 10­ 4 ppm F3,328 = 8.27, p < 0.001). Consumption increased signif- nicotine in sucrose solution was similar to the ­LD50 at cantly over time (Fig. 3b; F1,328 = 102.1, p < 0.001). The 2000 ppm reported by Detzel and Wink (1993). In compari- interactions for nicotine × colony (F3,330 = 0.36, p = 0.78) and son, dimethoate was 2000 times more toxic than nicotine nicotine × time (F3,329 = 0.81, p = 0.37) were not signifcant. (Table 4). Dimethoate is classifed as highly toxic to bees Nosema and nicotine doses in vivo (Q4) The young bees (i.e., LD­ 50 < 2 µg active ingredient per honey bee). With infected with Nosema and fed 10­ 4 ppm nicotine (in vivo respect to our frst research question (Q1), nicotine, at natu- experiment), consumed a mean of 16.2 μl sucrose solution ral levels found in nectar and pollen, is highly unlikely to over 2 days (Fig. 4a), with 0% bee survival over a 48 h period harm bees. (Fig. 4b). At a far lower nicotine dose (10­ 2 ppm), bees con- Nosema contributes to poor honey bee health and global sumed an average of 24.4 μl per day (Fig. 4a), and 80% of losses of honey bee colonies (Burnham 2019) and can the bees survived over 48 h (Fig. 4b)—although all of these reduce brood and honey production (Botías et al. 2013). bees were dead within 5 days. Bee consumption and survival Nosema infection may change bee behaviors, such as task 2 were correlated (R = 0.68, F1,6 = 12.5, p = 0.012). Midgut allocation (Goblirsch et al. 2013; Lecocq et al. 2016), Nosema spore counts 14 days post infection are shown with increase bee activity (Alaux et al. 2014; Wells et al. 2016), boxplots (Fig. 4c). The spore counts difered according to and cause infected bees to spend more time outside of their nicotine concentrations (χ2(5) = 13.1, p = 0.022), with a sig- colony but engage in shorter foraging bouts (Kralj and Fuchs nifcant diference between 1 and 100 ppm nicotine (Z = 3.0, 2010; Wolf et al. 2014; Dosselli et al. 2016). Regarding the

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H. P. Hendriksma et al. research question Q2, we found that bees steadily increased after a spore infects a bee midgut cell (Sin et al. 1997; Huang their sucrose consumption after experimental infection et al. 2013) to kill Nosema spores directly (Badowska- (Fig. 3b). Likewise, Naug and Gibbs (2009) found increased Czubik et al. 1984). Our pre-exposure of Nosema spores hunger in Nosema infected workers, and Mayack and Naug to nicotine at ­100, ­102 and ­104 ppm, before feeding them to (2010) found a decline in hemolymph sugar levels in bees, had no efect on Nosema virulence (Q5, Fig. 5). In con- Nosema infected foragers. Nosema lacks mitochondria and trast, the gut parasite Crithidia bombi, in a similar in vitro is, therefore, heavily dependent on the ATP production of its exposure experiment with nicotine, did slightly delay the host (Gómez-Moracho et al. 2017; Mayack and Naug 2009). development of this parasite after infecting its bumblebee The observed increased feeding may, therefore, be caused by host (Baracchi et al. 2015). One diference may be that C. decreased energy levels due to infection stress. bombi reproduces extracellularly in the gut lumen, whereas At low doses, nicotine may potentially enhance reward Nosema develops intracellularly, e.g., within epithelial cells, association in bees (Thany and Gauthier 2005; Gauthier where concentrations of imbibed nicotine are likely lower 2010; Stevenson et al. 2017), and foraging bumblebees than in the lumen. In addition, Nosema is a prokaryote and are evidently attracted to low nicotine levels (Manson Crithidia an eukaryote. Further, C. bombi lacks a rigid spore et al. 2010; Thorburn et al. 2015; Baracchi et al. 2017). At wall which protects Nosema spores from abiotic stressors the feld-realistic dose of 1.6 ppm, bumblebees were able such as heat and desiccation (Fenoy et al. 2009), and poten- to detect nicotine (Tiedeken et al. 2014). Our honey bees tially from compounds such as nicotine. infected with Nosema showed no preference for feeding on The methods used to feed bees Nosema spores can have 1 ppm versus 0 ppm nicotine in sucrose solution (Fig. 3a). variable efects on the fnal infection levels and on bee sur- However, the feld realistic dose of 1 ppm is relatively low vival (Milbrath et al. 2013; Urbieta-Magro et al. 2019). compared to nicotine concentrations that elicited honey bee Although we infected newly emerged bees and foragers attraction or repellence (Köhler et al. 2012; Singaravelan (Table 4) and used both group-level spore feeding (§2.4, et al. 2005, 2006). We note that nicotine can occur naturally §2.5) and individual bee feeding (§2.6), the fnal spore loads at concentrations higher than 1 ppm, i.e., 5 ppm in nectar were comparable between our experiments and were similar and 23 ppm in pollen (Tadmor-Melamed et al. 2004; Singa- to those found in other studies (Paxton et al. 2007; Maist- ravelan et al. 2005, 2006). Furthermore, the dose-dependent rello et al. 2008; Rubanov et al. 2019). We note that fresh deterrence of nicotine is stronger at a lower nectar concen- N. ceranae spores are thought to be relatively fragile and tration, such as 0.65 M sucrose (Köhler et al. 2012), but we susceptible to temperature stress (Fries 2010), hence the tested nicotine with a relative high concentration of 1.5 M preference for using fresh spore extracts for infection tri- sucrose solution that may have increased bee attraction and als. However, for the in vivo experiment, a three-week old decreased a repelling efect of nicotine. However, our results extract of N. ceranae maintained at 4 °C resulted in a 204- did not show that honey bees preferred nicotine solutions fold spore amplifcation rate (Table 4). Perhaps surprisingly, over controls (Fig. 3a), and infected bees did not increase given hypothesized spore fragility, in vitro pre-exposure to their uptake of sucrose solutions when nicotine concen- high nicotine solutions, and a subsequent water washing of trations were higher (Fig. 4a). This strongly suggests that spores, did not afect Nosema virulence (Table 4; 107-fold Nosema infection did not afect nicotine intake by honey and 315-fold amplifcation), as compared to unmanipulated bees (Q3). fresh spore extracts used for the choice and in vivo experi- Given that nicotine did also not actively suppress ments (Table 4; 142-fold and 204-fold, resp.). These results Nosema infection (Figs. 4c, 5), and merely increased mor- show that Nosema spores can be robust and remain virulent tality (Figs. 2a, 4b), it is not surprising that infected bees despite intense handling—as befts a widespread and com- did not prefer nicotine. Under no-choice feeding conditions mon pathogen. (Fig. 4), higher concentrations of nicotine (10­ 2, ­103 and Multiple studies have suggested that nicotine could help ­104 ppm) were aversive to bees. Similarly, bumblebees were honey bees or bumblebees fght of gut parasites (Köhler deterred by 50 ppm nicotine in sucrose solution (Baracchi et al. 2012; Baracchi et al. 2015; Biller et al. 2015; Richard- et al. 2017). Bee avoidance or rejection behavior may be son et al. 2015; Thorburn et al. 2015; Palmer-Young et al. explained by their gustatory ability to detect nicotine (De 2017a; De Roode and Hunter 2018). Our results show that Brito-Sanchez 2011), by higher-level efects based upon ace- dietary nicotine does not suppress N. ceranae infections in tylcholine receptor-based pathways (Gauthier 2010), or both. honey bees and bees did not exhibit self-medication. Yet Fumagillin pre-treatment of Nosema spores in vitro did honey bees, like many other insects, can be harmed by nico- not impair the ability of spores to infect bees (Fig. 5). This tine toxicity, via nicotinic acetylcholine receptor (nAChR) is perhaps not surprisingly given that the antibiotic fuma- activation, which promotes action potentials in postsynaptic gillin acts intracellularly by disrupting Nosema merogeny. nerve cells (Johnson 2015). Notwithstanding, other phyto- Fumagillin, therefore, afects Nosema in its vegetative stage, chemicals may suppress nosemosis and should be tested

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Nicotine does not reduce Nosema ceranae infection in honey bees given the importance of fnding new anti-microsporidian (2007) A metagenomic survey of microbes in honey bee colony agents (Holt and Grozinger 2016; Burnham 2019). collapse disorder. Science 318:283–287. https​://doi.org/10.1126/ scien​ce.11464​98 Acknowledgements Crane E, Walker P, Day P (1984) Directory of important world honey We are very grateful to the North American Pol- sources. International Bee Research Association, London, p 384 linator Protection Campaign (NAPPC) for their sponsoring via the Pol- de Brito-Sanchez MG (2011) Taste perception in honey bees. Chem linator Partnership; Award number: 20162891. We thank all researchers Senses 36(8):675–692. https​://doi.org/10.1093/chems​e/bjr04​0 from the Nieh lab (UCSD, San Diego, CA) and the Toth lab (ISU, de Roode JC, Hunter MD (2018) Self-medication in insects: when Ames, IA) for feedback on this study. 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Nicotine does not reduce Nosema ceranae infection in honey bees

in foral nectar reduce parasite infections in bumblebees. P R Soc Thany SH, Gauthier M (2005) Nicotine injected into the antennal lobes B 282:20142471. https​://doi.org/10.1098/rspb.2014.2471 induces a rapid modulation of sucrose threshold and improves Rubanov A, Russell KA, Rothman JA, Nieh JC, McFrederick QS short-term memory in the honeybee Apis mellifera. Brain Res (2019) Intensity of Nosema ceranae infection is associated 1039:216–219. https​://doi.org/10.1016/j.brain​res.2005.01.056 with specific honey bee gut bacteria and weakly associated Thorburn LP, Adler LS, Irwin RE, Palmer-Young EC (2015) Variable with gut microbiome structure. Sci Rep-UK 9:3820. https​://doi. efects of nicotine, anabasine, and their interactions on parasitized org/10.1038/s4159​8-019-40347​-6 bumble bees. F1000Res 4:880. https​://doi.org/10.12688​/f1000​ Schippers MP, Dukas R, Smith RW, Wang J, Smolen K, McClelland resea​rch.6870.2 GB (2006) Lifetime performance in foraging honeybees: behav- Tian B, Fadhil NH, Powell JE, Kwong WK, Moran NA (2012) Long- iour and physiology. J Exp Biol 209:3828–3836. https​://doi. term exposure to antibiotics has caused accumulation of resist- org/10.1242/jeb.02450​ ance determinants in the gut microbiota of honeybees. mBio Schneider-Orelli O (1947) Entomologisches Praktikum: Einführung 3(6):e00377-12. https​://doi.org/10.1128/mBio.00377​-12 in die land- und forstwirtschaftliche Insektenkunde. H.R. Sauer- Tiedeken EJ, Stout JC, Stevenson PC, Wright GA (2014) Bumblebees länder & Co., Aarau are not deterred by ecologically relevant concentrations of nec- Simone-Finstrom MD, Spivak M (2012) Increased resin collection after tar toxins. J Exp Biol 217:1620–1625. https​://doi.org/10.1242/ parasite challenge: a case of self-medication in honey bees? PLoS jeb.09754​3 One 7:e34601. https​://doi.org/10.1371/journ​al.pone.00346​01 Urbieta-Magro A, Higes M, Meana A, Barrios L, Martín-Hernández R Sin N, Meng L, Wang MQ, Wen JJ, Bornmann WG, Crews CM (1997) (2019) Age and method of inoculation infuence the infection of The anti-angiogenic agent fumagillin covalently binds and inhibits worker honey bees (Apis mellifera) by Nosema ceranae. Insects the methionine aminopeptidase, MetAP-2. PNAS 94:6099–6103. 10:417. https​://doi.org/10.3390/insec​ts101​20417​ https​://doi.org/10.1073/pnas.94.12.6099 Webster TC (1993) Nosema apis spore transmission among honey bees. Singaravelan N, Ne’eman G, Inbar M, Izhaki I (2005) Feeding Am Bee J 133:869–870 responses of free-flying honeybees to secondary compounds Wells T, Wolf S, Nicholls E, Groll H, Lim KS, Clark SJ, Swain mimicking foral nectars. J Chem Ecol 31:2791–2804. https​:// J, Osborne JL, Haughton AJ (2016) Flight performance doi.org/10.1007/s1088​6-005-8394-z of actively foraging honey bees is reduced by a common Singaravelan N, Inbar M, Ne’eman G, Distl M, Wink M, Izhaki I pathogen. Environ Microbiol Rep 8:728–737. https​://doi. (2006) The efects of nectar-nicotine on colony ftness of caged org/10.1111/1758-2229.12434​ honeybees. J Chem Ecol 32:49–59 Wolf S, McMahon DP, Lim KS, Pull CD, Clark SJ, Paxton RJ, Singer MS, Mace KC, Bernays EA (2009) Self-medication as adap- Osborne JL (2014) So near and yet so far: harmonic radar reveals tive plasticity: increased ingestion of plant toxins by parasitized reduced homing ability of nosema infected honeybees. PLoS One caterpillars. PLoS One 4:e4796. https​://doi.org/10.1371/journ​ 9:e103989. https​://doi.org/10.1371/journ​al.pone.01039​89 al.pone.00047​96 Wright GA, Baker DD, Palmer MJ, Stabler D, Mustard JA, Power Sinpoo C, Paxton RJ, Disayathanoowat T, Krongdang S, Chantawan- EF, Borland AM, Stevenson PC (2013) Cafeine in foral nectar nakul P (2018) Impact of Nosema ceranae and Nosema apis on enhances a pollinator’s memory of reward. Science 339:1202– individual worker bees of the two host species (Apis cerana and 1204. https​://doi.org/10.1126/scien​ce.12288​06 Apis mellifera) and regulation of host immune response. J Insect Yemor T (2016) Efects of propolis of Apis mellifera linneause, 1787 Physiol 105:1–8. https​://doi.org/10.1016/j.jinsp​hys.2017.12.010 and Trigona apicalis Smith, 1857 from diferent regions of Thai- Stevenson PC, Nicolson SW, Wright GA (2017) Plant secondary metab- land on experimental infection of Nosema ceranae in the asiatic olites in nectar: impacts on pollinators and ecological functions. honeybee Apis cerana Fabricius, 1793. Dissertation, Burapha Funct Ecol 31:65–75. https​://doi.org/10.1111/1365-2435.12761​ University Tadmor-Melamed H, Markman S, Arieli A, Distl M, Wink M, Izhaki Zander E (1909) Tierische Parasiten als Krankenheitserreger bei der I (2004) Limited ability of Palestine sunbirds Nectarinia osea Biene. Münchner Bienenzeitung 3:196–204 to cope with pyridine alkaloids in nectar of tree tobacco Nico- tiana glauca. Funct Ecol 18:844–850. https​://doi.org/10.111 1/j.0269-8463.2004.00929​.x

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